We have recently initiated an investigation of Insertion Sequence movements in multi drug resistant bacteria with a focus on carbapenemase-producing Enterobacteriacaea (CPE). Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, has been reported to be frequently associated with resistance determinants, but its role remained unclear. We have analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the NIH Clinical Center. We used target site duplications (TSDs) and their distribution patterns as guides and discovered that a large fraction of plasmid reorganizations result from IS26 intramolecular replicative transpositions, including replicon fusions, DNA inversions, and deletions. We have now extended these studies to other types of mobile elements. We are also interested in following the ongoing reorganization of plasmids carrying multidrug-resistant determinants from the perspective of their active mobile elements. More recently,resistance even for colistin, which is a last resort antibiotics have been observed to emerge in enterobacteriaceae. We have discovered that dissemination of the colistin resistance gene appears linked to a particular mobile genetic element, and we are in the process of its characterization. One prokaryotic transposition mechanism that clearly plays a central role in the emergence of multi antibiotic resistance is the so-call copy-out-paste-in mechanism. Despite its central importance, there is no current mechanistic information available regarding how this process works. Recently, we have identified a mobile genetic element called IS256 that uses this process and we have been able to generate crystals of the IS256 family transposase complexed with DNA that diffracts X-rays well. Crystallographic structure determination is underway. Curcio, M.J. and Derbyshire, K.M. (2003) Nat. Rev. Mol. Cell. Biol. 4, 865-877. Debets-Ossenkopp, Y.J., et al. (1999) Antimicrob. Agents Chemother. 43, 2657-2662. Sebaihia, M. et al. (2006) Nature Genet. 38, 779-786.